The overall goal of this proposal is to significantly expand on findings from our ongoing Merit award-supported work to further interrogate gastrointestinal tract colonization and disease establishment of the diarrheagenic pathogen Clostridium difficile. To enable this project, we have identified unique bacterial factors that mediate C. difficile interaction(s) with the host intestine. Three Specific Aims are proposed. First, we will expand on our studies defining C. difficile response to, and tolerance of, reactive oxygen species and redox stress. This response severely impacts GI tract colonization of the pathogen, and highly attenuated, potential vaccine strains were generated for these studies during the current award period. Second, we will continue studies to interrogate infectious mechanism(s) of the low-toxin/high-virulence C. difficile strains we recovered from our active disease surveillance of the Southern Arizona veteran and non-veteran population. The wealth of proteomic data we have garnered will be exploited for genetic studies to define virulence and colonization mechanisms of these strains. Third, and with the goal of translating our research into an immediately clinically-relevant context, we will validate and test our newly-engineered recombinant probiotic bacterium which is a targeted C. difficile colonization inhibitor. All studies in this proposal will use existing or t-be-collected specimens from the veteran population, and utilize both hamster and mouse models of acute C. difficile infection and colonization respectively. We will also incorporate state-of-the-at methodologies including next-generation omics and sequencing, and live-animal bioluminescence imaging to interrogate various aspects of C. difficile colonization and disease establishment.